Critical Role of Ca²⁺ Ions in the Reaction Mechanism of Euphorbia characias Peroxidase

Abstract

A cationic peroxidase was isolated and characterized from the latex of the perennial Mediterranean plant Euphorbia characias. The purified enzyme contained one heme prosthetic group identified as ferric iron-protoporphyrin IX. In addition, the purified peroxidase contained 1 mol of endogenous calcium per mol of enzyme; removal of this calcium ion resulted in almost complete loss of the enzyme activity. However, when excess Ca(2+) was added to the native enzyme the catalytic efficiency was enhanced by 3 orders of magnitude. The mechanism of activation was studied using a wide range of spectroscopic and analytic techniques. Analysis of the steady state by stopped-flow measurements suggests that the main effect of calcium ions is to favor the oxidation of the ferric enzyme by hydrogen peroxide to form compound I, whereas the other steps of the catalytic cycle seem to be affected to a lesser extent. UV/vis absorption spectra and CD measurements show that the heme iron is pentacoordinated high-spin in native enzyme and remains so after the binding of Ca(2+). Only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ca(2+) ions, except for a significant perturbation of the Fe(3+) inner sphere geometry, as detected by EPR measurements. We propose that Ca(2+) binding to a low affinity site induces a reorientation of the distal histidine changing the almost inactive form of Euphorbia peroxidase to a high activity form. This is the first example of a peroxidase that responds as an on/off switch to variations in the external Ca(2+) level.

Specific functions of individual class III peroxidase genes

“…Another interesting study concerns a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP; Medda et al, 2003). Calcium plays a central role as a second messenger in the regulation of a number of physiological processes in plants, as in all eukaryotes.…”

A probable crosstalk between Ca+2, reactive oxygen species accumulation and scavenging mechanisms and modulation of protein kinase C activity during seed development in sunflower

“…14 Present investigations aim at understanding the possible correlation among the various phases of seed development, generation, and enzymatic scavenging of ROS during these phases, activation of protein kinase C, and its modulation by calcium. In this context, qualitative and quantitative analysis of major ROS scavenging enzymes, namely superoxide dismutase (SOD), peroxidase (POD), and glutathione reductase (GR), has been undertaken at 3 stages of sunflower seed development (20,30, and 40 d after anthesis). Modulation of the activity of these enzymes with seed development has been examined for a possible correlation with calcium-stimulated protein kinase C (PKC) activity.…”

“…[19][20][21] Protein kinase C activity exhibits development-dependent modulation during seed maturation in sunflower. This has been observed both during early stages of embryo development and during the development of seeds by the use of PKC specific fluorescent probe Fim-1, a membrane permeant derivative of bisindoylmaleamide, as well as PKC inhibitors, namely staurosporine and bisindoylmaleamide.…”

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun-NN) plants over-expressing a synthetic HRP-C gene are altered in growth, development and susceptibility to abiotic stress

“…Such correlations are complicated by the large numbers of peroxidase genes that have been identified in a range of plant species [26,29,32,35], the expression of which is controlled by specific developmental and/or environmental signals [8,26,35]. Moreover, physiological control mechanisms affecting enzyme activity have been proposed that include calcium-mediated activation and de-activation reactions [22]. At present, the functional role of this high level of genetic and physiological redundancy is not clear.…”